The bestranking compounds from the three libraries was a popular hit within the LOPAC_3d library. The fitness scores are above
100.0 which means that ligands are most favorable for the inhibition assays.
Table 1. The top 12 compounds of Cb_306_3d.sdf, HF9PlatesPlates5_9.sdf, and LOPAC_3d.sdf libraries screened against MptpB active site.
Week 14: Enzyme Assays 12/01/11
Vary Substrate Phosphatase Enzyme Test
The data points form an increasing line. However,
there are a couple of points that are not positively
correlated to the rest of the data points.
Fig 7. MptpB enzyme assay with pNPP as surrogate substrate.
Phosphatase Enzyme Test
The data increases in an exponential regression as the concentration
increases. It is safe to continue to to the vary substrate enzyme test.
Fig 6. MptpB enzyme assay with MptpB as substrate (Trial 2).
Fig 5. Absorbance of MptpB measured at 410nm.
Phosphatase Enzyme Test
These are terrible data. The absorbance of the decreases as the concentration increases
on the interval [0,4.3] and then increases {4.3,9.8].
Fig 4. MptpB enzyme assay with MptpB as substrate (Trial 1).
Fig 3. Absorbance of MptpB measured at 410nm.
Fig 2. Concentration of concentrated MptpB to 0.5ml with 20% glycerol.
Fig 1. Concentration of elution 1 after concentrating MptpB to 0.5ml.
Week 13: FPLC Gel Filtration11/21/11
MptpB is possibly in peaks 42-47. The other three peaks are probably run-offs from the filtration job ran before
mine.
Week 12: Protein Characterization11/18-11/19
It is kind of hard to view gel, but there is a distinct band
for elution two.
From right to left: lane 1 is the PageRuler Ladder, lane 2 is cell lysate before induction, lane 3 is cell lysate after induction, lane 4 is soluble fraction, lane 5 is flow through, lane 6 is wash and lane 7 is elution 1, and lane 8 is elution 2.
Week 11: Protein Purification 11/09-11/10
Began with a protein pH of 7.0 which was sufficient for the BIO-RAD
chromophotography
Week 10: Protein Expression 11/02-11/04 •Proceeding to protein purification and characterization to check the efficacy of the expression
•If protein expression fails, IPTG concentration will be adjusted to 1mM and expressed for 6 hours at 30 oC to insure the activation of the lac promoter
Spectrophotometry Measurement
Absorbance=0.318 at 1.5 hours
Absorbance=0.869 at 2.0 hours
Transformation Plates
Week 10:Nanodrop Spectrophotometry 11/01/11
Finally obtained a great concentration of protein with 227.6 ng/ul.
Nanodrop of PCR squared (200ul) with a concentration of 227.6 ng/ul
Week 10:Gel Electrophoresis 10/31/11
In lane 7, I made a control secondary PCR composed of the leading
and lagging Mtptb primer to validate whether the PCR worked. In lane 5,
there were amplifications at around 1000 bp and below the DNA ladder.
I suspect that the secondary PCR worked this time because I made
new primers.
Lane1: 100BP DNA Ladder (5ul) Lane3: Primary PCR (15ul) Lane5: Secodary PCR (15ul) Lane7: Secondary PCR with leading and lagging Mptpb primers (15ul) Lane8: 1KB DNA Ladder (5ul) Lane9: pNIC-Bsa4 vector (10ul) Lane10: pNIC-Bsa4 vector (10ul)
Week 9:PCR Overlap Gel Electrophoresis 10/29/11
Not good at at all. The secondary PCR is being amplified about 100 BP which is
10 times below what was expected.
Ronnie - keep trying - you will get it. But let's start shifting the majority of your effort over to protein expression. - Dr. B
Lane1: 100 BP DNA Ladder Lane3: Secondary PCR (5th trial)
Week 9: pNIC-Bsa4 Gel Electrophoresis 10/28/11
This gel explains why my first two trials failed. The pNIC-Bsa4 were above the
1 KB DNA ladder. My next step is to wait 'til next week when Thou makes some
new pNIC-Bsa4. Lane1: 1 KB ladder (5ul) Lane3: pNIC-Bsa4 59.8 ng/ul (7ul) Lane5: pNIC-Bsa4 82.4 ng/ul (3ul) Lane7: 100 BP ladder (5ul)
Week 9:Virtual Screening 10/28/11
I was successfully able to identify why my best ranking values were
negative. The alpha helix of my pdb protein caused much torsional
strain against the library of inhibitors. Now I am using pdb 2OZ5,
which has an inhibitor already bound. Therefore, I can easily remove
the inhibitor and dock others. I am still in the process of troubleshooting
my runs and errors.
Fatal error:
licence request failed (for gold process creation):
Flexlm raised the following error: Licensed number of users already reached
Feature: gold
License path: /apps/gold/goldsuite-5.0/ccdc_licence.dat:/apps/gold -
/gold_v4.1/GOLD_Suite/ccdc_licence.dat:/apps/gold/gold_v4.0/GOLD_Suite -
/ccdc_licence.dat
FLEXlm error: -4,132
For further information, refer to the FLEXlm End User Manual,
available at "www.macrovision.com".
/apps/gold/goldsuite-5.0/ccdc_licence.dat
Week 8: pNIC-Bsa4 Gel Electrophoresis 10/20/11
I made a new pNIC-Bsa4 accepting vector.There were blue dye in all
of the lanes that were visible to the naked eye. I do not know why the
DNA Ladder was the only thing that showed up.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Secondary PCR (10ul) from primary PCR (1st Trial) Lane3: Tertiary PCR (10ul) from secondary PCR (1st Trial) Lane4: pNIC-Bsa4 vector (3.0ul)
Week 8:pNIC-Bsa4 Gel Electrophoresis 10/18/11 Nothing showed up for the pNIC-Bsa4 vector in lane 9. I used the NEB enzymes so next time the Fermentus enzymes instead.
Week 8:Protein Target Virtual Screen
I ran my GOLD run for the cb_306_3d and HF9library.
Week 7:Protein Target Virtual Screen
I ran my virtual screen Monday, but my run had a huge error file. Now my next
step is to rerun the first run.
Week 7:PCR Primer Overlap
Sixth Gel Electrophoresis 10/16/11
Same set-up as the fifth gel electrophoresis. It looks like
there is a band in lanes 3 and 5 approximately 800-1000 bp.
My best bet would probably be to PCR clean-up. It was
brought to my attention on 10/19 that I was using the protein
loading dye.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Skip Lane3: Secondary PCR (15ul) from primary PCR (1st Trial) Lane4: Skip Lane5: Tertiary PCR (15ul) from secondary PCR (1st Trial) Lane6: Skip Lane7: Secondary PCR (15ul) from primary PCR (4th Trial)
Fifth Gel Electrophoresis 10/14/11
This time, I figured I would use the primary and secondary PCR from the first trial
since my gel electrophoresis actually worked the first time. I made another secondary
PCR from the primary PCR (1st trial) and then a tertiary PCR from the secondary
PCR (1st trial). It was a failure so my next step is to repeat this step for verification.
This is very frustrated especially since I was like two weeks ahead of others,
and now they have caught up with me. My gels are still not coming out the
way I would like it to be. Hopefully, I will be able to finish the primer overlap
Sunday.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Secondary PCR (15ul) from primary PCR (1st Trial) Lane3: Skip Lane4: Tertiary PCR (15ul) from secondary PCR (1st Trial)
Fourth Gel Electrophoresis 10/14/11
Lane 7 is great, but it is the first trial that had a low concentration.
I cant figure out why the 2nd and 3rd trials are not working.I am doing the
same procedure.
Second Gel Electrophoresis 10/05/11
Evidently there was either an error in my protocol procedure or Joshua's MPTPb
primary PCR was no good. This graph should look like the first gel electrophoresis
with a distinct band at about 1000bp. The band shown at approximately 200bp
is probably just primers. I also found my PCR samples from the first gel electrophoresis,
but someone had left the PCR rack with my samples out at room temperature. My next
step would be to start the PCR overlap all the way from scratch since my PCR samples
might have denatured.
Ronnie - ok - good luck on getting it to work. -- Dr. B
Lane1: 100bp DNA ladder (5.0ul) Lane2: Secondary PCR (15ul) has a distinct band at about 200bp
Nanodrop Spectrophotometry 10/05/11
The data was definitely too low of an absorbance. I was expecting the peak to have an
absorbance close to 0.80nm.
Nanodrop Spectrophotometry 10/05/11
I wanted to double check my results. Changes implemented: cleaned the Nanodrop spectrophotomer
with ethanox, wiped with KimWipe, double blanked with water, pipetted 2ul of PCR-squared sample.
Even though I did not receive the desired absorbance, the quantity did increase by a factor of
1.56nm. In the future, I would take extra caution with disinfecting my environment and equipment,
and also reproofing my results or taking an average of trials experimented.
Third Gel Electrophoresis 10/06/11
Unfortunately, none of the PCRs are shown and the 100bp DNA Ladder is barely displayed.
I might just run another gel electrophoresis because I should have seen something.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Primary PCR (15ul) is shown as a smear Lane3: Secondary PCR (15ul) has a distinct band at about 1000bp
Week 5:PCR Primer Overlap -- Ronnie - good job. Don't need to post as much of the protocol - just focus more on your results like you show below in the image. Could label your gel lanes. -- Dr. B
Gene of Interest: MptpB Rv0153c
Purpose: Synthesize a gene of interest from scratch
Objective: To clone your assigned gene using overlap extension PCR with the oligos designed using DNAworks
Expression Vector Option B: pNIC-Bsa4 Continuing with expression vector option: You will go to ligation independent cloning (LIC) to insert the gene into the expression vector. Then you determine if it is correct by DNA sequencing.
Oligo Mix
Make a new microcentrifuge tube
Add autoclaved Nanopure water to the tube. The amount of water will need to be equal to 100 - # of primers you have
For Example, if you have 20 primers for your gene, you would need 80 ul of water.
Mix the contents of each primer in the deep well plate with a 200 ul pipette
Remove 1 ul of each stock primer from the plate and add to the new ‘Primer Mix’ tube
Primary PCR: 5 ul 10X rxn buffer 3 ul 25 mM MgSO4 5 ul 2 mM dNTPs 1 ul Template/Primers = 1 mM oligo mix 1 ul KOD hotstart Polymerase (1U/ml) 35 ul sterile dH2O 50 ul Final Volume
Secondary PCR: Thermocylcer cycling conditions similar as primary PCR but the number of cycles is increased to 30.
5 ul 10X rxn buffer 3 ul 25 mM MgSO4 5 ul 2 mM dNTPs 1 ul Template = primary PCR reaction (if more than one primary PCR reaction was set up, use 1ul from each primary PCR reaction) 1 ul 20 mM F primer (pNIC-Bsa4 custom primer 100 uM; 20 uM solution) 1 ul 20 mM R primer (pNIC-Bsa4 custom primer 100 uM; 20 uM solution) 1 ul KOD hotstart Polymerase (1U/ml) 33 ul sterile diH2O (adjust this volume if more than one primary PCR reaction was added to mix) 50 ul Final Volume
First Gel Electrophoresis 09/30/11
Some of the secondary PCR somehow have gotten into lane four which
would explain the similar band in lane 3.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Primary PCR (15ul) is shown as a smear Lane3: Secondary PCR (15ul) has a distinct band at about 1000bp
Week 4:Virtual Screening (Waiting on Second Run)
Week 4: PCR Primer Design for Primer Overlap Assembly PCR
Ronnie - show images instead of links to WORD docs - paste in your sequences for Primers here. . - Dr. B
Purpose: design an oligo set of forward and reverse primers for PCR synthesizing and amplifying the CDS of your gene of interest so that it can be inserted into a cloning (or expression) vector.
Go to the DNA Works website (http://helixweb.nih.gov/dnaworks/) to upload your sequence. This site will take the amino acid sequence and back translate it into DNA. When this step is performed, the DNA is also ‘codon optimized’ to match the most preferred codons of the organism in which you will express your construct (most likely E. coli BL21(DE3) cells).
Job Name: put a descriptive job title e.g. InitialsDateOrganismGeneName (EPB061611Ecolidhfr) E-mail Address: in put your email (highly recommended) Skip Mutant Run: Codon Frequency Table: select ‘E. coli class II’ Parameters: leave most as default except change ‘Oligo Length’ = 60 nt Skip Restriction Site Screen: Skip Custom Site Screen: Skip Weights: Sequences (s) (formats): select ‘protein’ Then paste your amino acid sequence into the ‘Choice 2 – Enter sequence manually’ It is recommended that you ‘Filter’ your protein sequence first by going to the Sequence Manipulation Suite and hitting ‘Filter Protein’ You need to be sure that a STOP amino acid is on the end (insert a ‘X’). Alternatively, we could add this in the amplification primers.
Week 4:Protocol of PCR for pNIC-Bsa4 cloning off of Cloning Vector
Steps
1) Select primers:
VDS15 For: CA2 Human, pNIC-Bsa4 (2.5 uM)
VDS16 Rev: CA2 Human, pNIC-Bsa4 (2.5 uM)
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube (ID:4190, 383.1 ng/ul)
5) Add water
6) Add MgSO4
7) Keep on ice
8) Preheat PCR machine
9) Add Taq polymerase dilution (2ul+8ul Nanopure)
10) Run PCR machine
11) Make Agarose Gel
First Gel Electrophoresis 09/23/11
Base pair 1400-1600 bp amplification was probably due to strong concentration of
primers, and not diluting. This gel electrophoresis was my best so far since I improved
my pipetting technique.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Sample A (12.5ul) Lane3: Sample B (12.5ul) Lane4: Sample C (12.5ul) Lane6: Sample D (12.5ul) Lane7: Sample E (12.5ul) Lane8: Sample F (12.5ul) Strong indicator of amplification at 800 bp and also at 1,517 bp.
Week 4:Virtual Screening (Started First Run)
Week 3: Pymol Refresher
Objective Examine three dimensional structure of a new enzyme
Background DHFR-TS from Trypanosoma cruzi is a bi-functional enzyme complex that carries out the role of dihydrofolate reductase and thymidylate synthase. T. cruzi is the pathogen responsible for Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. Potentially, this target could be used to inhibit growth of the parasite. 2H2Q3:08p.m. 09/15 This is the PDB identifier for the complex with the natural substrates. Make a PyMol image showing all of the components separately (each component should be selected individually and given a name). Display each chain distinctively. Show polar contacts between the protein and any substrates or cofactors.
Fig 1. Chain A of 2H2Q is represented distinctively by the substrate NAP (blue) shown as polar contacts to the active site indicated in (green) as sticks.
Fig 2. Chain B of 2H2Q is represented distinctively by the substrate NAP (yellow) shown as polar contacts to the active site indicated in (green) as sticks.
3CL9 5:36p.m. This is the PDB identifier for the A chain (DHFR portion) with a known inhibitor (MTX). Make an image showing all of the components and then the polar contacts between the inhibitor and protein. Highlight the active site (around 5 angstroms from ligand) in a different color. $select active, MTX around 5 #code to select active site around object ‘MTX’
Fig 3. MTX (yellow) is shown as sticks inhibiting the 3CL9 active site indicated by the polar contacts within 5 Angstroms.
Comparison to Human 1U72 6:21 p.m. This is the PDB identifier for Human DHFR with MTX. Create selections for all of the components. Then, highlight the active sites (around 5 angstroms from ligand) in different colors. Then, bring the two proteins together by performing an alignment to show how closely the T. cruzi and the H. sapiens structures line up (compare 1U72 and 3CL9). Record the RMS value. How close are the binding modes of MTX to each of these two enzymes? Do you think the enzymes could be differentially targeted with a single drug? Are there any differences in the amino acid sequence in the active site (i.e. amino acids that come in contact with the ligand)? To determine the active site, click on your active site selection and then copy down the amino acids that are highlighted in the Sequence Viewer above. Show a pairwise comparison of the active site sequences in your report. Are there any similarities/differences between them?
Fig 4. Representation of T. cruzi and the H. sapiens proteins superimposed with a RMS value=1.126. The MTX from 1U72 is shown as yellow by element, and the MTX from 3CL9 is shown as light blue by element. The MTX-active site interactions are magnified respectively in the two smaller images. The binding modes of the MTX are structurally identical except for a few variations within the aromatic rings and rotamers. The enzymes could be similarly targeted with a single drug since the MTX binding modes suggest a medium to high specificity towards the active site and probably a strong affinity.
Fig 5. Pairwise comparison of the active site sequences of 1U72 (yellow) and 3CL9 (green). One differnce in the amino acid sequence in the active site is serine in 1U72 and lysine in 3CL9.
3HBB 7:14 p.m. This is the PDB identifier for the complex with another known inhibitor (TMQ). Make a PyMol image showing all of the components and polar contacts between the protein and inhibitor. Display each chain distinctively. Highlight the active site (around 5 angstroms from ligand) in a different color. Is the binding mode of TMQ to T. cruzi DHFR-TS significantly different than that of MTX to human DHFR from 1U72? Address polar contacts and relevant amino acids in the active site.
Fig 6. TMQ (yellow) is shown as sticks inhibiting the 3CL9 active site indicated by the polar contacts within 5 Angstroms between oxygens and nitrogens. The binding mode of TMQ to T. cruzi DHFR-TS is significantly different than that of MTX to human DHFR from 1U72. The aromatic rings of TMQ are favorably hydrophobic and there are widely different conformational angles.
Ronnie - those are some pretty gels! Good job documenting your work. Good luck on the next attempt. - Dr. B
Week 2: PCR Protocol
Day 1
1) Make Template Dilutions
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube
5)Place on ice
6) Preheat PCR machine
7) Add Taq polymerase
8) Run PCR machine
Day 2
1) Make Agarose Gel
2) Run Gel for about 40 min
First Gel Electrophoresis 09/07/11
I used M13 forward and reverse primers.
Gel broke and lanes 3-6 did not show.
Lane1: 100bp DNA ladder + Blue Juice (7.0ul) Lane2: 100bp DNA ladder (5.0ul) Lane3: Sample A (12.0ul) Lane4: Sample B (12.0ul) Lane5: Sample C (12.0ul) Lane6: Sample D (12.0ul)
Restrictive Enzyme Digest
Day 1
1) Digestive Reactions
Eco RI
PvuII
EcoRI+PvuII
2) Make digestion in centrifuge tube
3) Incubate for 1-2 hours
4) Heat block
Day 2
1) Make Agarose Gel
2) Run gel for about 40-45 min
Second Gel Electrophoresis 09/09/11
This time I used SP6 promotor and T7 promoter primers.
The results were good, however there were probably
poor methods of administration. I'm speculating that
the samples did not settle into the wells correctly.
Lane1: 100bp DNA ladder (5.0ul) Lane2: Sample A (12.0ul) Lane3: Sample B (12.0ul) Lane4: Sample C (12.0ul) Lane6: Sample D (12.0ul) Lane7: Skip Lane8: 1kb DNA ladder (5.0ul) Lane9: Uncut Stock Plasmid pGBR22 (3.0 ul) Lane10: EcoRI RE Digestion Sample (25.0ul) Lane11: PvuII RE Digestion Sample (25.0ul) Lane12: EcoRI+PvuII RE Digestion Sample (25.0ul)
Protocol Submitting DNA to DNA Sequencing Facility
1) Place order for M13-For-20 (Order# 71971) online
2) Prepare template+M13-For primer+Nanopure
3) Deliver tube to DNA sequencing facility
Table 1. The table represents the distribution of 16 human nucleotide Blast Hits (National Library of Medicine) on the Query Sequence.
Nucleotide Collection
Table 2. The table represents the distribution of 158 Blast Hits (National Library of Medicine) on the Query Sequence for all genome sequences.
Purple Protein Coding Sequence Translation Results
Slicing and Dicing
The gray regions are the areas that were cut off by the restriction enzymes. Both of the restriction enzymes have 100% activity in NEB buffer.
Eco RI
Fig 1. The figure represents the origin of the restrictive enzyme EcoRI cutting the pGBR22 at a specific site.
PvuII
Fig 2. The figure represents two origins of the restrictive enzyme PvuII cutting the pGBR22 at specific sites.
PvuII+Eco RI
Fig 3. The figure represents three origins (one and two respectively) of the restrictive enzyme EcoRI + PvuII cutting the pGBR22 at specific sites.
Fig 3. The gel confirms that the restrictive enzymes will cut into distinguishable pieces. A reverse complement would be used to determine the sequence beyond the 1000 bp limit. Thus, we would be able to find the overlapping sequence, and add it to the 1000 bp already sequenced.
Ronnie's Research
Week 14: Virtual Screening 12/02/11
The bestranking compounds from the three libraries was a popular hit within the LOPAC_3d library. The fitness scores are above
100.0 which means that ligands are most favorable for the inhibition assays.
Week 14: Enzyme Assays 12/01/11
Vary Substrate Phosphatase Enzyme Test
The data points form an increasing line. However,
there are a couple of points that are not positively
correlated to the rest of the data points.
Phosphatase Enzyme Test
The data increases in an exponential regression as the concentration
increases. It is safe to continue to to the vary substrate enzyme test.
Phosphatase Enzyme Test
These are terrible data. The absorbance of the decreases as the concentration increases
on the interval [0,4.3] and then increases {4.3,9.8].
Week 13: FPLC Gel Filtration 11/21/11
MptpB is possibly in peaks 42-47. The other three peaks are probably run-offs from the filtration job ran before
mine.
Week 12: Protein Characterization 11/18-11/19
It is kind of hard to view gel, but there is a distinct band
for elution two.
Week 11: Protein Purification 11/09-11/10
Began with a protein pH of 7.0 which was sufficient for the BIO-RAD
chromophotography
Week 10: Protein Expression 11/02-11/04
•Proceeding to protein purification and characterization to check
the efficacy of the expression
•If protein expression fails, IPTG concentration will be adjusted to
1mM and expressed for 6 hours at 30 oC to insure the activation of
the lac promoter
Spectrophotometry Measurement
Absorbance=0.318 at 1.5 hours
Absorbance=0.869 at 2.0 hours
Transformation Plates
Week 10: Nanodrop Spectrophotometry 11/01/11
Finally obtained a great concentration of protein with 227.6 ng/ul.
Week 10: Gel Electrophoresis 10/31/11
In lane 7, I made a control secondary PCR composed of the leading
and lagging Mtptb primer to validate whether the PCR worked. In lane 5,
there were amplifications at around 1000 bp and below the DNA ladder.
I suspect that the secondary PCR worked this time because I made
new primers.
Week 9: PCR Overlap Gel Electrophoresis 10/29/11
Not good at at all. The secondary PCR is being amplified about 100 BP which is
10 times below what was expected.
Ronnie - keep trying - you will get it. But let's start shifting the majority of your effort over to protein expression. - Dr. B
Week 9: pNIC-Bsa4 Gel Electrophoresis 10/28/11
This gel explains why my first two trials failed. The pNIC-Bsa4 were above the
1 KB DNA ladder. My next step is to wait 'til next week when Thou makes some
new pNIC-Bsa4.
Lane1: 1 KB ladder (5ul)
Lane3: pNIC-Bsa4 59.8 ng/ul (7ul)
Lane5: pNIC-Bsa4 82.4 ng/ul (3ul)
Lane7: 100 BP ladder (5ul)
Week 9: Virtual Screening 10/28/11
I was successfully able to identify why my best ranking values were
negative. The alpha helix of my pdb protein caused much torsional
strain against the library of inhibitors. Now I am using pdb 2OZ5,
which has an inhibitor already bound. Therefore, I can easily remove
the inhibitor and dock others. I am still in the process of troubleshooting
my runs and errors.
Fatal error:
licence request failed (for gold process creation):
Flexlm raised the following error: Licensed number of users already reached
Feature: gold
License path: /apps/gold/goldsuite-5.0/ccdc_licence.dat:/apps/gold -
/gold_v4.1/GOLD_Suite/ccdc_licence.dat:/apps/gold/gold_v4.0/GOLD_Suite -
/ccdc_licence.dat
FLEXlm error: -4,132
For further information, refer to the FLEXlm End User Manual,
available at "www.macrovision.com".
/apps/gold/goldsuite-5.0/ccdc_licence.dat
Week 8: pNIC-Bsa4 Gel Electrophoresis 10/20/11
I made a new pNIC-Bsa4 accepting vector.There were blue dye in all
of the lanes that were visible to the naked eye. I do not know why the
DNA Ladder was the only thing that showed up.
Week 8: pNIC-Bsa4 Gel Electrophoresis 10/18/11
Nothing showed up for the pNIC-Bsa4 vector in lane 9. I used the NEB
enzymes so next time the Fermentus enzymes instead.
Week 8: Protein Target Virtual Screen
I ran my GOLD run for the cb_306_3d and HF9library.
Week 7: Protein Target Virtual Screen
I ran my virtual screen Monday, but my run had a huge error file. Now my next
step is to rerun the first run.
Week 7: PCR Primer Overlap
Sixth Gel Electrophoresis 10/16/11
Same set-up as the fifth gel electrophoresis. It looks like
there is a band in lanes 3 and 5 approximately 800-1000 bp.
My best bet would probably be to PCR clean-up. It was
brought to my attention on 10/19 that I was using the protein
loading dye.
Nanodrop Spectrophotometry 10/16/11
Secondary PCR (15ul) from primary PCR (1st Trial)
Nanodrop Spectrophotometry 10/16/11
Tertiary PCR (15ul) from secondary PCR (1st Trial)
Fifth Gel Electrophoresis 10/14/11
This time, I figured I would use the primary and secondary PCR from the first trial
since my gel electrophoresis actually worked the first time. I made another secondary
PCR from the primary PCR (1st trial) and then a tertiary PCR from the secondary
PCR (1st trial). It was a failure so my next step is to repeat this step for verification.
This is very frustrated especially since I was like two weeks ahead of others,
and now they have caught up with me. My gels are still not coming out the
way I would like it to be. Hopefully, I will be able to finish the primer overlap
Sunday.
Fourth Gel Electrophoresis 10/14/11
Lane 7 is great, but it is the first trial that had a low concentration.
I cant figure out why the 2nd and 3rd trials are not working.I am doing the
same procedure.
Third Gel Electrophoresis 10/14/11
Something went wrong with the gel. I'm guessing possibly cross-contamination.
Week 6: PCR Primer Overlap Continued
Second Gel Electrophoresis 10/05/11
Evidently there was either an error in my protocol procedure or Joshua's MPTPb
primary PCR was no good. This graph should look like the first gel electrophoresis
with a distinct band at about 1000bp. The band shown at approximately 200bp
is probably just primers. I also found my PCR samples from the first gel electrophoresis,
but someone had left the PCR rack with my samples out at room temperature. My next
step would be to start the PCR overlap all the way from scratch since my PCR samples
might have denatured.
Ronnie - ok - good luck on getting it to work. -- Dr. B
Nanodrop Spectrophotometry 10/05/11
The data was definitely too low of an absorbance. I was expecting the peak to have an
absorbance close to 0.80nm.
Nanodrop Spectrophotometry 10/05/11
I wanted to double check my results. Changes implemented: cleaned the Nanodrop spectrophotomer
with ethanox, wiped with KimWipe, double blanked with water, pipetted 2ul of PCR-squared sample.
Even though I did not receive the desired absorbance, the quantity did increase by a factor of
1.56nm. In the future, I would take extra caution with disinfecting my environment and equipment,
and also reproofing my results or taking an average of trials experimented.
Third Gel Electrophoresis 10/06/11
Unfortunately, none of the PCRs are shown and the 100bp DNA Ladder is barely displayed.
I might just run another gel electrophoresis because I should have seen something.
Week 6: PCR Primer Design for pNIC-Bsa4 Cloning
Nucleotide Blast
>emb|BX842572.1| Mycobacterium tuberculosis H37Rv complete genome; segment 1/13
Length=341957
Features in this part of subject sequence:
PHOSPHOTYROSINE PROTEIN PHOSPHATASE PTPB (PROTEIN-TYROSIN...
Score = 503 bits (272), Expect = 9e-139
Identities = 646/829 (78%), Gaps = 16/829 (2%)
Strand=Plus/Minus
Query 1 ATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTTCGTGATGTTGCGGACACCGCGACC 60
||||| || |||||||||||||| ||||||||||||||||| || || |||||||| |||
Sbjct 181985 ATGGCTGTCCGTGAACTGCCGGGCGCGTGGAACTTTCGTGACGTCGCCGACACCGCAACC 181926
Query 61 GCGCTGCGCCCTGGTCGTCTCTTCC-GTTCTTCTGAACTG-TCTCGCCTGGATGATGCCG 118
|| |||| || || || || |||| || | | || ||| | ||||| || || ||||
Sbjct 181925 GCATTGCGGCCGGGGCGGCTGTTCCGGTCCAGC-GAGCTGAGC-CGCCTCGACGACGCCG 181868
Query 119 GTCGTGCGACCCTCCGCCGTCTGGGTATCACCGATGTCGCGGACCTCCGCTCTTCTCGTG 178
| || || || || ||||| ||||| |||||||| || || ||||| || || || || |
Sbjct 181867 GCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGG 181808
Query 179 AAGTTGCACGTCGTGGTCCAGGCCGCGTCCCGGACGGTATCGACGTTCACCTGCTGCCGT 238
| ||||| || || |||||||| || || |||||||| |||||||| |||||||||||||
Sbjct 181807 AGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGT 181748
Query 239 TCCCGGATCTGGCTGATGACGACGCGGATGA--CAGCGCTCCGCATGAAACCGCGTTCAA 296
|||| || || || ||||| ||||| || || ||||| ||||| |||||||| |||||
Sbjct 181747 TCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCG--CCGCACGAAACCGCATTCAA 181690
Query 297 -ACGTCTGCTGACCAACGATGGCTCTAACGGTGAAAGCGGTGAAT-CTTCTCAGTCTATC 354
| | ||||| ||||| || || || ||||| || ||| |||| | | ||||| ||
Sbjct 181689 GA-GGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGC-CAGTCGATA 181632
Query 355 AACGACGCAGCGACGCGTTACATGACCGACGAATACCGCCAATTTCCGACCCGCAACGGT 414
|| ||||| || || || |||||||||||||| || |||||||| || || ||||| ||
Sbjct 181631 AATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGA 181572
Query 415 GCGCAACGTGCCCTGCATCGTGTAGTTACCCTGCTCGCGGCAGGCCGTCCAGTACTCACC 474
|| || || || || |||||||| || || ||||| || || || || || || ||||||
Sbjct 181571 GCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACC 181512
Query 475 CACTGTTTCGCGGGCAAAGACCGTACCGGTTTTGTGGTTGCGCTGGTTCTGGAAGCGGTT 534
||||| |||||||| || || || ||||| || ||||| |||||||| || ||||||||
Sbjct 181511 CACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTC 181452
Query 535 GGCCTCGATCGTGACGTTATCGTTGCAGACTACCTGCG-TTCTAATGACTCTGTTCCGCA 593
||||| || || ||||| ||||| || ||||||||||| | || ||||| || || ||
Sbjct 181451 GGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGC-AACGACTCCGTGCCACA 181393
Query 594 GCTCCGTGCGCGTATCTCTGAAATGATCCAGCAGCGTTTCGACACCGAACTCGCGCCTGA 653
|| || || || ||||| || ||||||||||||||||||||||||||||| || || ||
Sbjct 181392 ACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGA 181333
Query 654 AGTCGTTACCTTCACCAAAGCGCGTCTGAGCGACGGCGTTCTCGGTGTACGCGCTGAATA 713
|| || || |||||||| || || ||| |||||| || || ||||| ||||| || ||
Sbjct 181332 GGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTA 181273
Query 714 CCTGGCGGCTGCGCGTCAGACCATCGACGAAACGTACGGCTCTCTGGGTGGCTACCTCCG 773
|||||| || || || |||||||| ||||| || ||||| || ||||| |||||||| ||
Sbjct 181272 CCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCG 181213
Query 774 TGACGCGGGTATTT-CTCAGGCGACCGTTAATCGTATGCGTGGTGTGCT 821
||||| ||||| | ||||| || || || || ||||| || |||||
Sbjct 181212 CGACGCCGGTATCAGC-CAGGCCACAGTCAACCGGATGCGCGGGGTGCT 181165
Protein Blast
>lcl|15201 unnamed protein product
Length=276
Score = 549 bits (1415), Expect = 2e-161, Method: Compositional matrix adjust.
Identities = 276/276 (100%), Positives = 276/276 (100%), Gaps = 0/276 (0%)
Query 1 MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE 60
MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE
Sbjct 1 MAVRELPGAWNFRDVADTATALRPGRLFRSSELSRLDDAGRATLRRLGITDVADLRSSRE 60
Query 61 VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND 120
VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND
Sbjct 61 VARRGPGRVPDGIDVHLLPFPDLADDDADDSAPHETAFKRLLTNDGSNGESGESSQSIND 120
Query 121 AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL 180
AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL
Sbjct 121 AATRYMTDEYRQFPTRNGAQRALHRVVTLLAAGRPVLTHCFAGKDRTGFVVALVLEAVGL 180
Query 181 DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA 240
DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA
Sbjct 181 DRDVIVADYLRSNDSVPQLRARISEMIQQRFDTELAPEVVTFTKARLSDGVLGVRAEYLA 240
Query 241 AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG 276
AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG
Sbjct 241 AARQTIDETYGSLGGYLRDAGISQATVNRMRGVLLG 276
Week 6: Virtual Screening
Molecular Weight: 565.0582 [g/mol]
Molecular Formula: C25H25ClN2O7S2
XLogP3-AA: 4.6
H-Bond Donor: 3
H-Bond Acceptor: 9
Top 50 Bestranking
Week 5: PCR Primer Overlap -- Ronnie - good job. Don't need to post as much of the protocol - just focus more on your results like you show below in the image. Could label your gel lanes. -- Dr. B
Gene of Interest: MptpB Rv0153c
Purpose: Synthesize a gene of interest from scratch
Objective: To clone your assigned gene using overlap extension PCR with the oligos designed using DNAworks
Expression Vector Option B: pNIC-Bsa4
Continuing with expression vector option: You will go to ligation independent cloning (LIC) to insert the gene into the expression vector. Then you determine if it is correct by DNA sequencing.
Oligo Mix
Primary PCR:
5 ul 10X rxn buffer
3 ul 25 mM MgSO4
5 ul 2 mM dNTPs
1 ul Template/Primers = 1 mM oligo mix
1 ul KOD hotstart Polymerase (1U/ml)
35 ul sterile dH2O
50 ul Final Volume
Secondary PCR:
Thermocylcer cycling conditions similar as primary PCR but the number of cycles is increased to 30.
5 ul 10X rxn buffer
3 ul 25 mM MgSO4
5 ul 2 mM dNTPs
1 ul Template = primary PCR reaction (if more than one primary PCR reaction was set up, use 1ul from each primary PCR reaction)
1 ul 20 mM F primer (pNIC-Bsa4 custom primer 100 uM; 20 uM solution)
1 ul 20 mM R primer (pNIC-Bsa4 custom primer 100 uM; 20 uM solution)
1 ul KOD hotstart Polymerase (1U/ml)
33 ul sterile diH2O (adjust this volume if more than one primary PCR reaction was added to mix)
50 ul Final Volume
First Gel Electrophoresis 09/30/11
Some of the secondary PCR somehow have gotten into lane four which
would explain the similar band in lane 3.
Week 4: Virtual Screening (Waiting on Second Run)
Week 4: PCR Primer Design for Primer Overlap Assembly PCR
Ronnie - show images instead of links to WORD docs - paste in your sequences for Primers here. . - Dr. B
Purpose: design an oligo set of forward and reverse primers for PCR synthesizing and amplifying the CDS of your gene of interest so that it can be inserted into a cloning (or expression) vector.
Go to the DNA Works website (http://helixweb.nih.gov/dnaworks/) to upload your sequence. This site will take the amino acid sequence and back translate it into DNA. When this step is performed, the DNA is also ‘codon optimized’ to match the most preferred codons of the organism in which you will express your construct (most likely E. coli BL21(DE3) cells).
Job Name: put a descriptive job title e.g. InitialsDateOrganismGeneName (EPB061611Ecolidhfr)
E-mail Address: in put your email (highly recommended)
Skip Mutant Run:
Codon Frequency Table: select ‘E. coli class II’
Parameters: leave most as default except change ‘Oligo Length’ = 60 nt
Skip Restriction Site Screen:
Skip Custom Site Screen:
Skip Weights:
Sequences (s) (formats): select ‘protein’
Then paste your amino acid sequence into the ‘Choice 2 – Enter sequence manually’
It is recommended that you ‘Filter’ your protein sequence first by going to the Sequence Manipulation Suite and hitting ‘Filter Protein’
You need to be sure that a STOP amino acid is on the end (insert a ‘X’). Alternatively, we could add this in the amplification primers.
Hit ‘Design Oligos’
The DNA sequence # 1 is: ----------------------------------------------------------------
1 ATGGCGGTTCGTGAACTGCCGGGTGCGTGGAACTTTCGTGATGTTGCGGACACCGCGACC
61 GCGCTGCGCCCTGGTCGTCTCTTCCGTTCTTCTGAACTGTCTCGCCTGGATGATGCCGGT
121 CGTGCGACCCTCCGCCGTCTGGGTATCACCGATGTCGCGGACCTCCGCTCTTCTCGTGAA
181 GTTGCACGTCGTGGTCCAGGCCGCGTCCCGGACGGTATCGACGTTCACCTGCTGCCGTTC
241 CCGGATCTGGCTGATGACGACGCGGATGACAGCGCTCCGCATGAAACCGCGTTCAAACGT
301 CTGCTGACCAACGATGGCTCTAACGGTGAAAGCGGTGAATCTTCTCAGTCTATCAACGAC
361 GCAGCGACGCGTTACATGACCGACGAATACCGCCAATTTCCGACCCGCAACGGTGCGCAA
421 CGTGCCCTGCATCGTGTAGTTACCCTGCTCGCGGCAGGCCGTCCAGTACTCACCCACTGT
481 TTCGCGGGCAAAGACCGTACCGGTTTTGTGGTTGCGCTGGTTCTGGAAGCGGTTGGCCTC
541 GATCGTGACGTTATCGTTGCAGACTACCTGCGTTCTAATGACTCTGTTCCGCAGCTCCGT
601 GCGCGTATCTCTGAAATGATCCAGCAGCGTTTCGACACCGAACTCGCGCCTGAAGTCGTT
661 ACCTTCACCAAAGCGCGTCTGAGCGACGGCGTTCTCGGTGTACGCGCTGAATACCTGGCG
721 GCTGCGCGTCAGACCATCGACGAAACGTACGGCTCTCTGGGTGGCTACCTCCGTGACGCG
781 GGTATTTCTCAGGCGACCGTTAATCGTATGCGTGGTGTGCTCCTGGGTTAA
Week 4: Protocol of PCR for pNIC-Bsa4 cloning off of Cloning Vector
Steps
1) Select primers:
VDS15 For: CA2 Human, pNIC-Bsa4 (2.5 uM)
VDS16 Rev: CA2 Human, pNIC-Bsa4 (2.5 uM)
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube (ID:4190, 383.1 ng/ul)
5) Add water
6) Add MgSO4
7) Keep on ice
8) Preheat PCR machine
9) Add Taq polymerase dilution (2ul+8ul Nanopure)
10) Run PCR machine
11) Make Agarose Gel
First Gel Electrophoresis 09/23/11
Base pair 1400-1600 bp amplification was probably due to strong concentration of
primers, and not diluting. This gel electrophoresis was my best so far since I improved
my pipetting technique.
Week 4: Virtual Screening (Started First Run)
Week 3: Pymol Refresher
Objective
Examine three dimensional structure of a new enzyme
Background
DHFR-TS from Trypanosoma cruzi is a bi-functional enzyme complex that carries out the role of dihydrofolate reductase and thymidylate synthase. T. cruzi is the pathogen responsible for Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. Potentially, this target could be used to inhibit growth of the parasite.
2H2Q 3:08p.m. 09/15
This is the PDB identifier for the complex with the natural substrates. Make a PyMol image showing all of the components separately (each component should be selected individually and given a name). Display each chain distinctively. Show polar contacts between the protein and any substrates or cofactors.
3CL9 5:36p.m.
This is the PDB identifier for the A chain (DHFR portion) with a known inhibitor (MTX). Make an image showing all of the components and then the polar contacts between the inhibitor and protein. Highlight the active site (around 5 angstroms from ligand) in a different color.
$select active, MTX around 5 #code to select active site around object ‘MTX’
Comparison to Human
1U72 6:21 p.m.
This is the PDB identifier for Human DHFR with MTX. Create selections for all of the components. Then, highlight the active sites (around 5 angstroms from ligand) in different colors. Then, bring the two proteins together by performing an alignment to show how closely the T. cruzi and the H. sapiens structures line up (compare 1U72 and 3CL9). Record the RMS value. How close are the binding modes of MTX to each of these two enzymes? Do you think the enzymes could be differentially targeted with a single drug? Are there any differences in the amino acid sequence in the active site (i.e. amino acids that come in contact with the ligand)? To determine the active site, click on your active site selection and then copy down the amino acids that are highlighted in the Sequence Viewer above. Show a pairwise comparison of the active site sequences in your report. Are there any similarities/differences between them?
Fig 5. Pairwise comparison of the active site sequences of 1U72 (yellow) and 3CL9 (green). One differnce in the amino acid sequence in the active site is serine in 1U72 and lysine in 3CL9.
3HBB 7:14 p.m.
This is the PDB identifier for the complex with another known inhibitor (TMQ). Make a PyMol image showing all of the components and polar contacts between the protein and inhibitor. Display each chain distinctively. Highlight the active site (around 5 angstroms from ligand) in a different color. Is the binding mode of TMQ to T. cruzi DHFR-TS significantly different than that of MTX to human DHFR from 1U72? Address polar contacts and relevant amino acids in the active site.
Ronnie - those are some pretty gels! Good job documenting your work. Good luck on the next attempt. - Dr. B
Week 2: PCR Protocol
Day 1
1) Make Template Dilutions
2) Make master mix
3) Add master mix to each tube
4) Add DNA template to each tube
5)Place on ice
6) Preheat PCR machine
7) Add Taq polymerase
8) Run PCR machine
Day 2
1) Make Agarose Gel
2) Run Gel for about 40 min
First Gel Electrophoresis 09/07/11
I used M13 forward and reverse primers.
Gel broke and lanes 3-6 did not show.
Restrictive Enzyme Digest
Day 1
1) Digestive Reactions
- Eco RI
- PvuII
- EcoRI+PvuII
2) Make digestion in centrifuge tube3) Incubate for 1-2 hours
4) Heat block
Day 2
1) Make Agarose Gel
2) Run gel for about 40-45 min
Second Gel Electrophoresis 09/09/11
This time I used SP6 promotor and T7 promoter primers.
The results were good, however there were probably
poor methods of administration. I'm speculating that
the samples did not settle into the wells correctly.
Protocol Submitting DNA to DNA Sequencing Facility
1) Place order for M13-For-20 (Order# 71971) online
2) Prepare template+M13-For primer+Nanopure
3) Deliver tube to DNA sequencing facility
Week 1: Analyzing DNA Sequence of Plasmid
Objective-Determine the DNA sequence of a plasmid
M13R DNA Sequence
TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGG
AGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGAT
GGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGG
AAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAG
TGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGC
ACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGA
ATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGA
GCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAAT
TCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCT
TTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCN
GGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
Nucleotide Human Blast Human G+T (4 databases)
Nucleotide Collection
Purple Protein Coding Sequence Translation Results
Slicing and Dicing
The gray regions are the areas that were cut off by the restriction enzymes. Both of the restriction enzymes have 100% activity in NEB buffer.